Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Take a look at some of the hottest news in life science these days: Tissue engineering, embryonic stem cells, cancer therapeutics—the public just can’t get enough of these topics, and rightly so. They impact human health.
Yet underlying these flashy subjects is perhaps the most mundane of cell-biology chores: cell culture. Somebody needs to tend and feed those cells, split them when they get crowded and expand them when it’s time to do an experiment. Where, though, do the methods for doing those culture tasks come from?
For well-studied cell lines, culture conditions are generally well established, and lines obtained from cell banks usually come with protocols for maintaining them. That’s not to say they can’t be made better. Ditto for cells serving as biological factories; in that case, researchers may be looking to maximize production of, say, therapeutic antibodies. And what about new cell lines? Researchers need to work those methods out for themselves.
The process can be exceedingly tedious. There are literally dozens of variables that come into play in cell culture, and optimizing all of them could take months to years. Here, Biocompare reviews a few tools and services that can help researchers accomplish these tasks.
Extracellular matrix
Adherent cells don’t generally stick to cell-culture dishes directly. They adhere to a thin layer of extracellular matrix (ECM) protein. Many such proteins exist, and deciding which to use is not always obvious.
EMD Millipore’s ECM Cell Culture Optimization Array enables researchers to quickly work out ideal ECM conditions for their particular cell line. According to product documentation, the kit contains a 96-well plate in which each of four ECM proteins, collagen I, laminin, fibronectin and vitronectin, are arrayed in triplicate at six concentrations from 20 ug/ml to 0.125 ug/ml. (The remaining wells include BSA and an uncoated negative control.)
The assay is takes just a few hours and is read colorimetrically. Cells are incubated in the wells to allow them to adhere, after which the wells are washed and stained to quantify how well the cells stuck to the ECM.
Media optimization
As cells grow, they consume the nutrients in their growth medium and release by-products. This can be especially problematic for bioprocess development, in which the goal is maximizing protein production.
One approach is to quantify metabolites in the growth medium over time, to identify compounds that are either limiting or overly abundant. In 2011, for instance, researchers at Bristol-Myers Squibb and MIT used “metabolic flux analysis” to optimize the growth and protein production of a continuous-growth culture of Chinese hamster ovary (CHO) cells in semi-steady state growth [1].
The team’s analysis, which considered some 32 metabolic parameters, “indicated that methionine, tryptophan, asparagine and serine were limiting, while alanine, arginine, glutamine and glycine were in excess at the semi-steady states.” Adjusting those amino acid levels—that is, reducing the levels of the excess amino acids in the growth medium and adding more of the limiting ones—boosted cell density by about half and improved protein concentration by a quarter, the authors reported.
Spinnovation Biologics, based in the Netherlands, offers a Spent Media Analysis service based on the company’s Spedia-NMR™ technology. Using nuclear magnetic resonance, the service simultaneously quantifies 20 to 50 culture components, including unwanted contaminants like ammonia.
In 2011, Spinnovation chief executive officer Frederic Girard wrote a tutorial about the service for Genetic Engineering & Biotechnology News. It includes a case study in which the company used Spedia-NMR to optimize CHO cell-based protein production for one of its clients. Analysis of culture media “showed a rapid increase in both formate and lactate levels and a rapid decrease in the level of the amino acid asparagine,” Girard wrote.
The asparagine, it turns out, was being degraded to ammonia, while the elevated lactate levels were dampening protein production – conditions the client was able to mitigate through altered culture conditions, Girard wrote.
Bioreactor optimization
In academic labs, cells are grown mostly in culture dishes. But as cell culture scales up to the bioprocess level, T-flasks just can’t cut it, and neither do the growth conditions that were used in them. As a result, researchers looking to scale up to bioreactors need to work out their culture conditions first. Several companies have developed small-scale bioreactor systems to facilitate this process.
TAP Biosystems’ ambr™ microscale bioreactor miniaturizes bioprocess development down to the 10 to 15 ml scale with an automated workstation that can control 24 or 48 reactors in parallel. Each reactor contains a small “impeller” that mixes and aerates the culture, just as in a larger bioreactor, and atmospheric conditions can be controlled on a per-reactor basis.
According to the ambr product page, the “ambr micro bioreactor system offers an accurate and cost-effective approach to replicating bioreactor conditions, and can be used as a microscale model for a wide range of upstream processes such as cell line selection and process optimisation.”
Also making relatively small-scale bioreactors is DasGip™, now part of Eppendorf. A 2012 article describes the use of 250-ml DasGip bioreactors to optimize stem cell growth in suspension [2].
Even with such tools, optimization takes time. You can outsource the work, though. A number of companies (and university core facilities) offer cell-line optimization services, including EMD Millipore, Life Technologies, Xcellerex (part of GE Healthcare Life Sciences) and Sophion.
Bottom line: Cell biology is nothing without cells. With tools like these, there’s no reason your cell cultures can’t be running at peak efficiency.
References
[1] Xing, Z, Kenty, B, Koyrakh, I, Borys, M, Pan, S-H, Li, ZJ, “Optimizing amino acid composition of CHO cell culture media for a fusion protein production,” Process Biochemistry, 46(7):1423-29, 2011.
[2] Olmer, R, Selzer, S, Zweigerdt, R, “Massively Expanding Stem Cell Suspensions: Achieving Optimal Cultivation and Maintaining Pluripotency and Differentiation Potential Counts,” Genetic Engineering & Biotechnology News, 32(20):32-33, 2012.
[3] Girard, F, “Cell Culture Media Optimization: NMR-Based Assay Method Seeks to Improve the Productivity of Biologics R&D and Processing,” Genetic Engineering & Biotechnology News, 31(21): 46-47, 2011.